Identification of potential miRNAs related to classification and treatment response of actinic keratosis

The Workflow activity was the following: Preliminary phase: Identification of 18 Formalin-fixed paraffin embedded (FFPE) samples (9 patients) («matched» 9 AK lesions and 9 SCC lesions). Working on biopsies samples we perform an extraction and RNA analysis with droplet Digital PCR (ddPCR) and we perform the data analysis. Second and final step phase: Evaluation of additional 39 subjects (36 men and 3 women). Results: We perform an evaluation and comparison of the following miRNA: miR-320 (a miRNA involved in apoptosis and cell proliferation control; miR-204, a miRNA involved in cell proliferation in and miRNA-16-5p, a miRNA involved in apoptosis).Conclusion: Our data suggest that there is no significant variation in the expression of the three tested microRNAs between adjacent AK lesions and squamous-cell carcinoma. However, a relevant trend has been observed Furthermore, by evaluating the miRNA expression trend between keratosis and carcinoma of the same patient, it is observed that there is no "uniform trend": for some samples the expression rises for the transition from AK to SCC and viceversa.

Diffusing alpha particles radiation treatment optimization in clinical settings

Alpha-particle emitters, notably used in 224Ra-DaRT, have emerged as effective in overcoming radiation resistance and providing targeted cancer therapy. These emitters cause DNA double-strand breaks, visualizable in human lymphocytes. The 224Ra DaRT technique, using a decay chain from seeds, extends alpha particle range, achieving complete tumor destruction while sparing healthy tissue. This thesis examines a biokinetic model, validated with patient data, and a feasibility study on skin squamous cell carcinomas are discussed. The study reports 75% tumor complete response rate and 48% patients experiencing acute grade 2 toxicity, resolving within a month. An observed abscopal effect (AE), where tumor regression occurs at non-irradiated sites, is examined, highlighting DaRT's potential in triggering anti-tumor immune responses. This effect, coupled with DaRT's high-linear energy transfer (LET), suggests its superiority over low-LET radiation in certain clinical scenarios. Improvements to DaRT, including the use of an external radio-opaque template for treatment planning, are explored. This advancement aids in determining source numbers for optimal tumor coverage, enhancing DaRT’s safety. The thesis outlines a typical DaRT procedure, from tumor measurements to source assessment and administration, emphasizing the importance of precise seed positioning. Furthermore, the thesis discusses DaRT's potential in treating prostate cancer, a prevalent global health issue, by offering an alternative to traditional salvage therapies. DaRT seeds, delivering alpha particle-based interstitial radiation, require precision in seed insertion due to their limited tissue range. In conclusion, the thesis advocates for DaRT's role in treating solid tumors, emphasizing its improved radiobiological potency and potential benefits over beta and gamma source-based therapies. Ongoing studies are assessing DaRT's feasibility in treating various solid tumors, including pancreatic, breast, prostate, and vulvar malignancies, suggesting a promising future in cancer treatment.

Progressione dalla cheratosi attinica al carcinoma squamocellulare localmente avanzato: clinica, dermatoscopia e marker molecolari

Il carcinoma a cellule squamose è un tumore della pelle la cui incidenza è in costante crescita. Per questo motivo si sta ritagliando uno spazio sempre più importante all’interno di quella che è la dermatologia oncologica. Sebbene la nostra accuratezza diagnostica sia in progressivo miglioramento rimangono due nodi fondamentali da sciogliere: la differenziazione delle forme precoci dalla controparte precancerosa (cheratosi attinica), ed il riconoscimento di lesioni particolarmente aggressive con possibile prognosi infausta per stabilire un trattamento adeguato. La maggior attenzione rivolta a queste neoplasie ha portato negli ultimi anni ad innumerevoli pubblicazioni ed alla produzione di molteplici linee guida con indicazioni talvolta non conclusive, che spesso creano confusione nella pratica clinica quotidiana. In questo studio vengono prese in esame queste due problematiche analizzando la casistica a nostra disposizione. Vengono quindi valutati i criteri diagnostici dermoscopici ed il follow-up clinico e strumentale del carcinoma a cellule squamose con un intento di semplificare per rendere più agevole la pratica clinica. Inoltre, viene valutata l’utilità di alcuni marker molecolari come le proteine p16 e Ki67, che risultano facilmente reperibili, e la cui ricerca risulta poco costosa per valutarne l’utilità di uno studio più ampio in occasione di migliorare la definizione prognostica di queste lesioni.

Bevacizumab and erlotinib (BE) first-line therapy in advanced non-squamous non-small-cell lung cancer (NSCLC) (stage IIIB/IV) followed by platinum-based chemotherapy (CT) at disease progression: A multicenter phase II trial (SAKK 19/05)

This phase II trial aimed to evaluate feasibility and efficacy of a first-line combination of targeted therapies for advanced non-squamous NSCLC: bevacizumab (B) and erlotinib (E), followed by platinum-based CT at disease progression (PD).

Initiation of Assembly of the Cell Envelope Barrier Structure of Stratified Squamous Epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.

HLA-G polymorphisms in women with squamous intraepithelial lesions harboring human papillomavirus

Human papillomavirus (HPV) infection is etiologically associated with low-(LSIL) and high-grade squamous intraepithelial lesions (HSIL) and with cervical cancer. The progression or regression of the lesions may depend, among other factors, on the host heritable immune response. Because human leukocyte antigen (HLA)-G molecules are involved in the modulation of innate and adaptive immune responses, and because no previous studies have evaluated HLA-G polymorphism in patients with SIL, we conducted a study to assess the association between HLA-G polymorphisms and cervical lesions harboring HPV infection. Cervico-vaginal scrapings and blood samples were collected from 125 women with SIL (68 LSIL and 57 HSIL) and from 94 healthy women without HPV infection and cytological abnormalities. HPV type and HLA-G polymorphisms in exons 2, 3 and 8 (14 bp insertion/deletion) were evaluated by PCR methodology, and digested with restriction endonucleases. The Genepop software and the EM and PHASE algorithms were used for statistical analysis. A significant protective association was observed between the presence of the G*0103 allele and SIL and between the G0101/G0104 genotype and HSIL in the group of patients compared to control. The presence of the G0104/+14 bp and G0104/-14 bp haplotypes conferred susceptibility to SIL compared to control. In addition, patients possessing the G0104/+14 bp haplotype and harboring HPV-16 and -18 co-infections were particularly associated with HSIL. These findings suggest that HLA-G polymorphisms may be associated with HPV infection and SIL, consequently representing a profile of predisposition to cervical cancer. Modern Pathology (2009) 22, 1075-1082; doi: 10.1038/modpathol.2009.67; published online 1 May 2009

Low level expression of human papillomavirus type 16 (HPV16) E6 in squamous epithelium does not elicit E6 specific B- or T-helper immunological responses, or influence the outcome of immunisation with E6 protein

Mice transgenic for E6/E7 oncogenes of Human Papillomavirus type 16 display life-long expression of E6 in lens and skin epithelium, and develop inflammatory skin disease late in life, which progresses to papillomata and squamous carcinoma in some mice. We asked whether endogenous expression of E6 induced a specific immunological outcome, i.e. immunity or tolerance, or whether the mice remained immunologically naive to E6. We show that prior to the onset of skin disease, E6 transgenic mice did not develop a spontaneous E6-directed antibody response, nor did they display T-cell proliferative responses to dominant T-helper epitope peptides within E6. In contrast, old mice in which skin disease had arisen, developed antibodies to E6. We also show that following immunisation with E6, specific antibody responses did not differ significantly among groups of EB-transgenic mice of different ages (and therefore of different durations and amounts of exposure to endogenous E6), and non-transgenic controls. Additionally, E6 immunisation-induced T-cell proliferative responses were similar in E6-transgenic and non-transgenic mice. These data are consistent with the interpretation that unimmunised Eb-transgenic mice that have not developed inflammatory skin disease remain immunologically naive to E6 at the B- and Th levels. There are implications for E6-mediated tumorigenesis in humans, and for the development of putative E6 therapeutic vaccines. (C) 2001 Elsevier Science B.V. All rights reserved.

Manipulating keratinocyte stem cell proliferation and differentiation using RNA interference

ABSTRACT : The epidermis, the outermost compartment of the skin, is a stratified and squamous epithelium that constantly self-renews. Keratinocytes, which represent the main epidermal population, are responsible for its cohesion and barrier function. Epidermal renewal necessitates a fine equilibrium between keratinocyte proliferation and differentiation. The keratinocyte stem cell, located in the basal cell layer, is responsible for epidermal homeostasis and regeneration during the wound healing process. The transcription factor p63 structurally belongs to the p53 superfamily. It is expressed in the basal and supra-basal cell layers of stratified epithelia and is thought to be important for the renewal or the differentiation of keratinocyte stem cells (Yang et al., 1999; Mills et al., 1999). In order to better understand its function, we established an in vitro model of p63 deficient human keratinocyte stem cells using a shp63 mediated RNA interference. Knockdown of endogenous p63 induces downregulation of cell-adhesion genes as previously described (Carroll et al., 2006). Interestingly, the replating of attached p63-knockdown keratinocytes on a feeder layer results in a loss of attachment and proliferation. They are no longer clonogenic. However, if the same population are replated in a fibrin matrix, extended fibrinolysis is reported, a common process in wound healing, suggesting that p63 regulates the fibrinolytic pathway. This result was confirmed by Q-PCR and shows that the urokinase pathway, which mediates fibrinolysis, is upregulated. Altogether, these findings suggest a mechanism in which the fine tuning of p63 expression promotes attachment or release of the keratinocyte stem cell from the basement membrane by inducing genes of adhesion and/or of fibrinolysis. This mechanism may be important for epidermal self-renewal, differentiation as well as wound healing. Its misregulation may be partly responsible for the p63 knockout phenotype. The downregulation of p63 also induces a decrease in LEKTI expression. LEKTI (lymphoepithelial Kazal-type serine protease inhibitor) is a serine protease inhibitor encoded by the Spink5 gene. It is expressed and secreted in the uppermost differentiated layers of stratified epithelia and plays a role in the desquamation process. When this gene is disrupted, humans develop the Netherton syndrome (Chavanas et al., 2000b). It is a dermatosis characterized by hair dysplasias, ichtyosiform erythroderma and impairment in epidermal barrier function promoting inflammation similarly as in psoriasis with inflammatory infiltrate in excess. TNFα (tumor necrosis factor alpha) and EDA1 (ectodysplasin A1) are two transmembraneprecursors that belong to the TNF superfamily, which is involved in immune and inflammation regulation (Smahi et al., 2002). We suggest that the secreted serine protease inhibitor LEKTI plays a role in the regulation of TNFα and EDA1 precursor cleavage and absence of LEKTI induces excess of inflammation. To investigate this hypothesis, we induced downregulation of Spink5 expression in rat keratinocyte stem cells by using a shSpink5 mediated RNA interference approach. Interestingly, expression of TNFα and EDA1 is modified after knockdown of Spink5 by Q-PCR. Moreover, downregulation of Spink5 induces loss of cohesiveness between keratinocytes and colonies adopt a scattered phenotype. Altogether, these preliminary data suggest that downregulation of LEKTI may play a role in the inflammatory response in Netherton syndrome patients, by regulating TNFα expression.